Modification of Egg Plasma Membrane

描述（由申请人提供）：在利用外部受精的物种中，包括海胆、青蛙和鱼类等模型系统，都表现出快速的蛋白酪氨酸激酶（PTK）激活，这是精子诱导的钙过渡、减数分裂II的退出、原核会议和有丝分裂所需的关键步骤。最近的研究表明，哺乳动物卵子在控制精子诱导的钙瞬变的机制上与低等动物有所不同。然而，尽管对小鼠和大鼠卵母细胞的研究表明，src家族激酶活性并不需要钙信号传导，但有强有力的证据表明，这些激酶在Mil恢复和原核合成中发挥作用。本提案的主要目标是确定哪些src家族激酶是对MM恢复和原核形成至关重要的信号通路。第一个具体目标将检验单个Src家族PTKs （Fyn， Yes或Src）是恢复减数分裂和原核会聚所必需的假设。实验方法将使用src家族敲除小鼠和显性阴性结构的组合来确定每种激酶在这些过程中的作用。第二个目标将确定这些激酶参与完成每个功能的特定生化途径。在这里，生化分析将确定哪些途径被每个激酶Fyn、Yes和Src的抑制所中断。将建立各途径与MM恢复原核会议的关系。第三个具体目标将检验Fyn， Yes和Src在受精后受到不同调节的假设，并且它们的活性在卵子激活期间的特定点增加，从而使它们能够控制MM恢复和原核会议。这一目的还将确定这些激酶在受精卵中被激活的调节机制。本研究的结果将提供对调节MM恢复和原核会聚的生化机制的理解，并将导致未来的研究使我们能够人为地调节这些过程。受精需要激活多种酶才能使卵子发育成胚胎。这项研究将使用更灵敏的现代技术来确定哪些蛋白激酶会触发受精卵排出额外的染色体，并将男性和女性的细胞核移动到一起。

DESCRIPTION (provided by applicant): Fertilization in species that utilize external fertilization, including model systems such as sea urchin, frog, and fish, all exhibit rapid protein tyrosine kinase (PTK) activation as a critical step required for the sperm- induced calcium transient, exit from meiosis II, pronuclear congression, and mitosis. Recent studies have demonstrated that mammalian eggs differ from lower species as to the mechanism controlling the sperm- induced calcium transient. However, while studies in oocytes of mice and rats indicate that Src-family kinase activity is not required for calcium signaling, there is strong evidence that these kinases play a role in Mil resumption and pronuclear congression. The main goals of this proposal are to establish which Src-family kinases are required signaling pathways critical to MM resumption and pronuclear congression. The first specific aim will test the hypothesis that individual Src-family PTKs (Fyn, Yes, or Src) are required for resumption of meiosis and pronuclear congression. The experimental approach will use a combination of Src-family knockout mice and dominant-negative constructs to define the role of each kinase in these processes. The Second aim will identify the specific biochemical pathways in which these kinases participate to accomplish each function. Here biochemical analyses will establish which pathways have been interrupted by suppression of each individual kinase Fyn, Yes, and Src. The relationship of each pathway to MM resumption of pronuclear congression will be established. The third specific aim will test the hypothesis that Fyn, Yes, and Src are differentially regulated after fertilization and that their activity increases at specific points during egg activation that allow them to control MM resumption and pronuclear congression. This aim will also identify the regulatory mechanisms by which these kinases are activated in the fertilized egg. The results of this study will provide an understanding of the biochemical mechanisms regulating MM resumption and pronuclear congression and will lead to future studies enabling us to modulate these processes artificially. Lay Summary: Fertilization requires that multiple enzymes become turned on in order for the egg to grow into an embryo. This study will use more sensitive, modern techniques to establish which protein kinases trigger the fertilized egg to eject extra chromosomes and move the male and female nuclei together.